Simultaneous determination of ethanol's four types of non-oxidative metabolites in human whole blood by liquid chromatography tandem mass spectrometry

- Ten ethanol non-oxidative metabolites in human whole blood with a wide range of polarity were analyzed simultaneously.
- Small sample consumption (50 μL) was used, making it suitable in cases with limited specimens.
- Different extraction procedures (liquid-liquid extraction, solid-phase extraction and protein precipitation) were compared.
- The proposed method was applied to two authentic samples, in which the metabolites provided critical information.

The importance of ethanol non-oxidative metabolites as the specific biomarkers of alcohol consumption in clinical and forensic settings is increasingly acknowledged. Simultaneous determination of these metabolites can provide a wealth of information like drinking habit and history, but it was difficult to achieve because of their wide range of polarity. This work describes development and validation of a simple liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for 4 types of ethanol non-oxidative metabolites (ethyl glucuronide, ethyl sulfate, fatty acid ethyl esters and phosphatidylethanols) in 50 μL of human whole blood. Pretreatment method, column and MS conditions were optimized. For the first time, the four types of ethanol non-oxidative metabolites with enormous discrepancies of property were simultaneously extracted and analyzed in one run within 40 min. The limits of detections (LODs) were among 0.1-10 ng/mL, and good linearity was obtained. Deviations in precision and accuracy were all lower than 15% at three QC levels. This method was then applied to two forensic samples, resulting in information on drinking habits and drinking time which were very useful for the interpretation of the blood alcohol results.

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